US20230047040A1 - Composition for treating neurodegenerative disorders comprising mesenchymal stem cells - Google Patents

Composition for treating neurodegenerative disorders comprising mesenchymal stem cells Download PDF

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US20230047040A1
US20230047040A1 US17/758,509 US202117758509A US2023047040A1 US 20230047040 A1 US20230047040 A1 US 20230047040A1 US 202117758509 A US202117758509 A US 202117758509A US 2023047040 A1 US2023047040 A1 US 2023047040A1
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cerebellum
stem cells
neurodegenerative diseases
treating neurodegenerative
cells
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Kyung Suk Kim
Tae Yong Lee
Kyoung Ho Suk
Ho Won Lee
Sang Ryong Kim
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Corestem Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the present invention relates to a method or a composition for treating neurodegenerative diseases, and particularly, the present invention relates to a method for treating neurodegenerative diseases of the cerebellum, comprising administering a composition comprising mesenchymal stem cells as an active ingredient to a patient having a neurodegenerative disease of the cerebellum, or to a composition for treating neurodegenerative diseases of the cerebellum comprising mesenchymal stem cells as an active ingredient.
  • Cerebellar ataxia and multiple system atrophy are motor neurodegenerative diseases caused by pathological processes affecting the cerebellum or related pathways. Cerebellar ataxia and multiple system atrophy are characterized by abnormal coordination and imbalance of movements, such as gait disturbance.
  • Cerebellar ataxia and multiple system atrophy can be caused by genetic defects, sporadic neurodegeneration disorders, acquired diseases (for example, infection, toxic reaction, alcohol, and vitamin deficiency), and other unknown causes.
  • acquired diseases for example, infection, toxic reaction, alcohol, and vitamin deficiency
  • cerebellar inflammation may play an important role in the progression of cerebellar ataxia and multiple system atrophy.
  • neurotoxic inflammatory responses involving the production of inflammatory molecules by activation of microglia and astrocytes have been observed in patients and animal models of hereditary cerebellar ataxia, particularly spinocerebellar ataxia.
  • Cerebellar inflammation is known to induce the loss of Purkinje cells, which results in cerebellar dysfunction.
  • the cerebellum is particularly lethal to poisons and poisoning, and acquired cerebellar ataxia and multiple system atrophy may occur due to inflammatory responses caused by viral infection and poison exposure (alcohol, drug, and environmental toxicity).
  • cerebellar ataxia and multiple system atrophy The fundamental mechanism of neurodegenerative diseases including cerebellar ataxia and multiple system atrophy is the gradual appearance of functional and quantitative damage to nerve cells.
  • neurodegenerative diseases such as cerebellar ataxia and multiple system atrophy cannot be prevented or treated, and only in extremely exceptional cases, treatment or symptom relief is possible.
  • mesenchymal stem cells are attracting attention as therapeutic candidates for neurodegenerative diseases because of their anti-inflammatory function, regenerative ability, and low immunoreactivity.
  • Human mesenchymal stem cells hMSCs
  • hMSCs are multipotent cells having the ability to self-regenerate and differentiate into several specialized cells.
  • hMSCs selectively target the site of damage and secrete various growth factors, cytokines, and chemokines known to have a wide range of activities, such as anti-apoptosis, angiogenesis, anti-inflammation, immunomodulation, and chemoattraction.
  • hMSCs are known to have therapeutic effects on neurological diseases such as Alzheimer's disease, Parkinson's disease, and stroke, there is little information on the therapeutic effect of mesenchymal stem cells in patients and animal models of a neurodegenerative disease of the cerebellum.
  • the present inventors established an animal model by directly injecting lipopolyssacharide (LPS) into the cerebellum or by injecting Ara-C (cytarabine) into the abdominal cavity in order to confirm the effect of hMSCs in neurodegenerative diseases of the cerebellum including cerebellar ataxia and multiple system atrophy.
  • LPS lipopolyssacharide
  • Ara-C cytarabine
  • the present inventors tried to confirm the therapeutic effect of human mesenchymal stem cells (hMSCs) in the three animal models including the SCA2 disease animal model, which is a genetic animal model through genetic modification.
  • An object of the present invention is to provide a method for treating neurodegenerative diseases of the cerebellum, comprising administering a composition comprising stem cells as an active ingredient to a patient having a neurodegenerative disease of the cerebellum.
  • Another object of the present invention is to provide a composition for alleviating or treating symptoms of neurodegenerative diseases of the cerebellum, comprising stem cells as an active ingredient.
  • the present invention provides a method for treating neurodegenerative diseases of the cerebellum, comprising administering a composition comprising stem cells as an active ingredient to a patient having a neurodegenerative disease of the cerebellum, or a composition for alleviating or treating symptoms of neurodegenerative diseases of the cerebellum, comprising stem cells as an active ingredient.
  • the present invention established an animal model of a neurodegenerative disease by administering LPS to the animal model to induce inflammation in the cerebellum, or by administering Ara-C to inhibit the development of the cerebellum, and constructed an animal model with SCA2 genetic modification disease that is genetically stabilized through the fifth generation.
  • MSCs mesenchymal stem cells
  • the term “neurodegenerative disease of the cerebellum” refers to a neurological disease in which motion is clumsy and there is no coordination between motions due to abnormal functions of the cerebellum, and includes all neurodegenerative diseases induced by various medical and neurological diseases or genetic predisposition.
  • the neurodegenerative disease of the cerebellum includes a neurodegenerative disease induced by inflammation, a neurodegenerative disease induced by toxins, or a neurodegenerative disease caused by genetic modification.
  • the neurodegenerative disease of the cerebellum includes cerebellar ataxia or multiple system atrophy.
  • stem cell refers to an undifferentiation cell having the ability to differentiate into various body tissues, and more specifically, may include an undifferentiated cell, which has the ability to differentiate into any specific or a plurality of functional cells and the self-replicating ability to repeatedly produce the same cells themselves. It is generated in all tissues during the development of a fetus, and may be found in some tissues where cells are actively replaced, such as bone marrow and epithelial tissue, even in adulthood.
  • the stem cell of the present invention may be an adult stem cell, and the adult stem cell may be at least one selected from the group consisting of a mesenchymal stem cell (MSC), a mesenchymal stromal cell, and a multipotent stem cell, but is not limited thereto.
  • MSC mesenchymal stem cell
  • a mesenchymal stromal cell a mesenchymal stromal cell
  • a multipotent stem cell but is not limited thereto.
  • the stem cell of the present invention may be a human mesenchymal stem cell (hMSC), but is not limited thereto.
  • hMSC human mesenchymal stem cell
  • mesenchymal stem cell refers to a stem cell (multipotent stem cell) that has multipotency before being differentiated into a cell of a specific organ such as bone, cartilage, fat, nerve tissue, fibroblast, and a muscle cell.
  • the mesenchymal stem cell may be preferably derived from one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnionic membrane, chorion, decidua, and placenta, but is not limited thereto.
  • an allogeneic bone marrow-derived mesenchymal stem cell was used as the stem cell.
  • bone marrow which is donated from normal and healthy donors by a method of collecting and isolating bone marrow, is diluted 1:2 with CSBM-A06 medium and then instilled on the Ficoll layer of the same amount of bone marrow prepared in advance. It is centrifuged at 400 ⁇ g for 30 minutes, and the obtained monocyte cell layer may be isolated, washed twice with CSBM-A06 medium, and then cultured under culture conditions in which 37° C. and 5% CO 2 is maintained. After 48 hours of culture, cells not attached to the bottom of the flask are removed by exchanging a new medium, and cells attached to the bottom are cultured by changing the medium once every 3 to 4 days. When the cultured cells grew to about 80%, they were subcultured into a new flask, and some of the cultured cells were continuously subcultured, and some were suspended in a cryopreservation solution containing DMSO and stored frozen.
  • a cell culture medium containing 10% FBS may be used as a culture medium usable for isolation and culture of stem cells.
  • a cell culture medium any cell culture medium commonly used in the art, such as Dulbecco's modified Eagle medium (DMEM), minimal essential medium (MEM), alpha-minimal essential medium (a-MEM), McCoys 5A medium, Eagle's basal medium, CMRL (Connaught Medical Research Laboratory) medium, Glasgow's minimal essential medium, Ham's F-12 medium, IMDM (Iscove's modified Dulbecco's medium), Liebovitz' L-15 medium, and RPMI (Roswell Park Memorial Institute) 1640 medium, may be used.
  • DMEM Dulbecco's modified Eagle medium
  • MEM minimal essential medium
  • a-MEM alpha-minimal essential medium
  • McCoys 5A medium Eagle's basal medium
  • CMRL Connaught Medical Research Laboratory
  • Glasgow's minimal essential medium Ham's F-12 medium
  • IMDM I
  • the stem cells according to the present invention express a mesenchymal stem cell-positive surface marker including CD29, CD44, CD73, CD90 or CD105, and 5% or less, preferably 3% or less of the stem cells according to the present invention express a negative surface marker including CD34 or CD45.
  • the stem cells according to the present invention when directly transplanted into the cisterna magna of an animal model with a neurodegenerative disease, may induce anti-inflammatory activity of the cerebellum and reduce the expression of inflammatory cytokines (IL-1 ⁇ or TNF ⁇ ) or inflammatory chemokines (MIP-1 ⁇ or MCP-1).
  • the stem cells according to the present invention may inhibit the activation of M1-type microglia, which are inflammatory neuroglia, and activate M2-type microglia, which are anti-inflammatory neuroglia, thereby inducing anti-inflammatory activity of the cerebellum.
  • the stem cells according to the present invention inhibit damage of Purkinje cell, and thus may be usefully used to alleviate or treat abnormal behavioral motor disorder symptoms.
  • the composition of the present invention may further contain one or more known active ingredients having a therapeutic effect of neurodegenerative diseases such as multiple system atrophy together with the stem cells.
  • composition of the present invention may include an appropriate suspending agent commonly used in the preparation of therapeutic compositions.
  • an injection may further include a preservative, an analgesic agent, a solubilizer or a stabilizer, etc.
  • a formulation for topical administration may further include a base, an excipient, a lubricant, or a preservative, etc.
  • the term “administration” means providing a given composition of the present invention to a subject by any appropriate method.
  • the dosage of stem cells is 2.0 ⁇ 10 4 to 1.0 ⁇ 10 6 cells/animal, and may be administered once or divided into several times for single administration or repeated administration.
  • the actual dosage of the active ingredient should be determined in view of several related factors such as the disease to be treated, the severity of the disease, the route of administration, the subject's body weight, age, and sex, etc. Therefore, the dosage is not intended to limit the scope of the present invention in any aspect.
  • composition of the present invention may be in the form of an injection.
  • composition of the present invention may be administered to a subject by various routes.
  • the administration may be intrathecal, intravenous, intramuscular, intraarterial, intramedullary, intradural, intraneural, intraventricular (subventricular zone), intracerebrovascular administration, and the like, and preferably, it may be directly transplanted into the dura mater of a subject in need of treatment, but is not limited thereto.
  • treatment comprehensively refers to improving symptoms of a disease related to a neurodegenerative disease of the cerebellum. It may include curing (bringing the condition substantially identical to that of a normal subject) or substantially preventing (inhibiting or delaying the onset of the disease) such a disease, or alleviating the condition thereof (ameliorating or beneficially altering the condition), and may include alleviating, curing or preventing one symptom or most of symptoms resulting from a disease related to cerebellar ataxia and multiple system atrophy, but is not limited thereto.
  • the present invention provides a method for preparing an animal model of a neurodegenerative disease of the cerebellum, comprising injecting an inflammation-inducing substance into the cerebellum of an animal other than a human, and an animal model of the disease prepared using the same.
  • the inflammation-inducing substance may be LPS (lipopolysaccharide).
  • the animal model was prepared by directly injecting LPS into the cerebellum of a mouse using a syringe.
  • it was prepared by directly injecting LPS at a concentration of 5 ⁇ g/5 ⁇ l into the cerebellum of a mouse, but the concentration and injection amount of each substance and the generation may be regulated depending on the species, age, and weight of an animal model to be prepared, and the like, within the technical knowledge of one of ordinary skill in the art.
  • the animal model of a neurodegenerative disease induced by an inflammation-inducing substance is one in which the inflammatory response limited to the cerebellum is activated by directly administering LPS to the cerebellum, and ataxia symptoms due to dysfunction and damage of Purkinje cells observed in a patient and a animal model of cerebellar ataxia in an inflammatory environment are shown.
  • the activation of M1-type microglia was induced after the LPS injection, and a neuroinflammatory response of the cerebellum was induced by increasing the expression of pro-inflammatory cytokines such as IL-1 ⁇ and TNF ⁇ or pro-inflammatory chemokines such as MIP-1 and MCP-1.
  • pro-inflammatory cytokines such as IL-1 ⁇ and TNF ⁇
  • pro-inflammatory chemokines such as MIP-1 and MCP-1.
  • damage or apoptosis of Purkinje cells was induced after the LPS injection.
  • the present invention provides a method for preparing an animal model of a neurodegenerative disease of the cerebellum, comprising directly injecting an anti-miotic agent into the cerebellum of an animal other than a human, and an animal model of the disease prepared using the same.
  • the anti-miotic agent may be Ara-C (cytarabine).
  • the animal model was prepared by directly injecting Ara-C into the abdominal cavity of a mouse using a syringe.
  • it was prepared by injecting 40 ⁇ g/kg of Ara-C into the abdominal cavity of an animal of 1-3 days of age, but the concentration and injection amount of each substance and the generation may be regulated depending on the species, age, and weight of an animal model to be prepared, and the like, within the technical knowledge of one of ordinary skill in the art.
  • the animal model of a neurodegenerative disease induced by an anti-miotic agent is one in which the development of the cerebellum is inhibited and artificial ataxia occurs by directly administering Ara-C into the abdominal cavity of an animal continuously (once a day, a total of 3 times) between day 1 and day 3 after birth, before cerebellar development progresses.
  • cerebellar development occurs after 14 days of age. Purkinje cells are observed at embryo day 13, and an external granule layer, a Purkinje cell layer, and an internal granule layer of the cerebellum are formed after birth.
  • the cerebellum it is the only organ that develops after birth, and by directly administering an anti-miotic agent at this time, a cerebellum maturation disorder may be induced and a neurodegenerative disease may be induced.
  • the present invention provides a method for preparing an animal model of a genetic neurodegenerative disease, comprising overexpressing or inhibiting a gene of an animal other than a human, and an animal model of the disease prepared using the same.
  • the overexpressed gene may be SCA2.
  • the genetic animal model utilizes an animal model with a genetically stabilized disease through repeated backcrossing with a C57BL/6J mouse up to the fifth generation, but the concentration and injection amount of each substance and the generation may be regulated depending on the species, age, and weight of an animal model to be prepared, and the like, within the technical knowledge of one of ordinary skill in the art.
  • the animal model of a neurodegenerative disease caused by genetic modification is a B6D2-Tg(Pcp2-SCA2)11Plt/J mouse in which the human SCA2 gene is inserted and overexpressed into the Pcp2 promoter, and an animal model in which abnormal neurological symptoms such as ataxia and staggering appear from week 8 and about 50% of Purkinje cells are lost at week 24 to week 27 may be used.
  • the treatment method or the treatment composition using mesenchymal stem cells, according to the present invention has remarkable effects in reducing neuroinflammation, inhibiting M1 microglia, activating M2 microglia, inhibiting apoptosis of Purkinje cells, inhibiting death of neurons, improving motor ability, and the like, and therefore may be effectively utilized in alleviating and treating neurodegenerative diseases including cerebellar ataxia and multiple system atrophy.
  • FIG. 1 A shows a result obtained by confirming the construction of an animal model of a disease induced by LPS administration. It is a result obtained by confirming the expression levels of Iba1 (microglia), inflammatory cytokines (IL-1 ⁇ and TNF ⁇ ), and inflammatory chemokines (MCP-1 and MIP-1 ⁇ ) in the cerebellum on day 1 and day 7 after LPS administration.
  • Iba1 microglia
  • MCP-1 and MIP-1 ⁇ inflammatory chemokines
  • FIG. 1 B shows a result obtained by confirming the construction of an animal model of a disease induced by LPS administration. It is a result obtained by confirming the expression level of Purkinje cells in the cerebellum and rotarod test for 4 weeks after LPS administration, and it is a result obtained by confirming the establishment of an animal model of a neurodegenerative disease induced by inflammation.
  • FIG. 3 A shows a result obtained by establishing an animal model of a disease in which genetic stability is secured by backcrossing up to the fifth generation, which is a neurodegenerative disease SCA2 animal model through genetic modification.
  • FIG. 3 B shows a result obtained by performing the rotarod test to evaluate the behavioral motor of a neurodegenerative disease SCA2 animal model through genetic modification of the fifth generation.
  • FIG. 4 A shows a result obtained by observing the spindle-shaped morphology of the initial cultured hMSCs (within P5) by microscope.
  • FIG. 4 C shows a result obtained by analyzing the immunophenotyping for the expression profile of the cell surface markers of early stage hMSCs.
  • FIG. 4 D shows a result obtained by confirming the multilineage differentiation ability of hMSCs.
  • FIG. 4 E shows a result obtained by confirming the neurotropic factors expressed in hMSCs in the culture solution, and it is a result obtained by confirming the expression of ANG, BDNF, and IGF, which are well known neuroprotective and growth factors.
  • FIG. 5 A shows a result obtained by performing the rotarod test for 4 weeks after transplanting hMSCs into a mouse in which the cerebellar inflammatory response is induced by LPS.
  • FIG. 5 C shows a result obtained by confirming the expression level of Iba1 (microglia-related) in the cerebellum exposed to LPS at 7 days after transplantation of hMSCs.
  • *p ⁇ 0.05 vs. CON, # p ⁇ 0.05 and ## p ⁇ 0.01 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n 3).
  • FIG. 5 D shows a result obtained by confirming the expression level of pro-inflammatory cytokines (IL- 1 ⁇ and TNF ⁇ ) in the cerebellum at 7 days after transplantation of hMSCs.
  • IL-1 ⁇ **p ⁇ 0.01 vs. CON, ## p ⁇ 0.01 and ### p ⁇ 0.001 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n 3).
  • FIG. 5 F shows a result obtained by confirming the expression level of CD86 and CD206 in the cerebellum at 7 days after transplantation of hMSCs.
  • FIG. 6 A shows a result obtained by confirming the effect of improving motor ability according to the concentration and frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C (rotarod test).
  • FIG. 6 C shows a result obtained by comparing the effect of improving general motor activity according to the concentration and frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C.
  • *p ⁇ 0.05 vs. WT, # p ⁇ 0.05 and ## p ⁇ 0.01 vs. Ara-C (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n 3).
  • FIG. 6 E shows a result obtained by confirming the improvement of neuromotor disorder according to the dose and frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C.
  • ***p ⁇ 0.001 vs. WT, # p ⁇ 0.05 vs. Ara-C (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n 3).
  • FIG. 7 shows a result obtained by confirming the effect of improving motor ability according to the dose of administration of hMSCs in an animal model of a SCA2 genetic disease caused by genetic modification.
  • mice Male C57BL/6 mice (Daehan Biolink, Republic of Korea) and B6D2-Tg(Pcp2-SCA2)11Plt/J mice were bred in a controlled environment with a 12-hour photoperiod, and food was distributed ad libitum. All animal experimental procedures were performed in accordance with the regulations of the Animal Experimental Ethics Committee of Kyungpook National University (No. KNU 2016-42).
  • Inflammation-related cerebellar ataxia was induced by directly injecting lipopolyssacharide (LPS, 5 ⁇ g/5 ⁇ L) into the cerebellum of mice.
  • LPS lipopolyssacharide
  • 10-week-old mice were anesthetized by injecting 115 mg/kg of ketamine (Yuhan, Republic of Korea) and 23 mg/kg of Rompun (Bayer Korea, Republic of Korea) into the abdominal cavity of 10-week-old mice, and then fixed on a stereotaxic device (David Kopf Instruments, Tujunga, Calif., USA).
  • a stereotaxic device David Kopf Instruments, Tujunga, Calif., USA.
  • the skull was exposed through a central sagittal incision, and then burr hole opening was performed.
  • LPS LPS
  • PBS phosphate buffered saline
  • the head of the mouse injected with LPS was turned about 90° toward the body in the stereotaxic device.
  • the underlying dura mater was exposed, and then hMSCs (1 ⁇ 10 5 cells/20 ⁇ L or 1 ⁇ 10 6 cells/20 ⁇ L) were transplanted into the cisterna magna using a Hamilton syringe (25 ⁇ L, 30 G) connected to an injection pump.
  • the needle was removed after 10 minutes, and the incision site was sutured with a silk suture.
  • hMSCs Human Mesenchymal Stem Cells
  • Ara-C an anti-miotic agent
  • a cerebellar ataxia independent of inflammation in mice.
  • an animal model of neurotoxicity induced cerebellar ataxia was constructed by intraperitoneal administration of Ara-C at a concentration of 40 mg/kg daily to mice from day 1 to day 3 after birth.
  • hMSCs (1 ⁇ 10 5 cells/20 ⁇ L or 1 ⁇ 10 6 cells/20 ⁇ L) were transplanted into the cisterna magna of the mice injected with Ara-C.
  • Mononuclear cells were isolated from the bone marrow by density gradient centrifugation using Ficoll (Ficoll-Paque Premium; GE Healthcare Bio-Sciences AB), plated in CSBM-A06 medium (Corestem, Republic of Korea) at a density of about 1 ⁇ 10 5 cells/cm 2 , and then cultured with 10% FBS (fetal bovine serum; Life Technologies, Grand Island, N.Y., USA), 2.5 mM L-alanyl-L-glutamine (Biochrom AG, Berlin, Germany), and 1% penicillin-streptomycin (Biochrom AG). Cells not attached were removed with a fresh medium, and then the medium was changed once every 3 to 4 days. Cells grown to 70-80% confluency were defined as passage 0 (passage zero, P0). Before subsequent experiments, cells were subcultured until passage 10 (P10).
  • Ficoll Ficoll-Paque Premium
  • GE Healthcare Bio-Sciences AB Ficoll-Paque Premium
  • PTD Population doubling time
  • T0 is the cell transplantation time
  • T is the cell harvest time
  • N0 is the initial number of cells
  • N is the number of harvested cells.
  • Multilineage differentiation assays Cell differentiation was induced according to the prior art. Specifically, hMSCs were cultured in a 24-well plate and stimulated to be differentiated according to adipogenic lineage, osteogenic lineage, and chondrogenic lineage using an hMSC functional identification kit (R&D Systems, Minneapolis, Minn., USA). After differentiation, fat droplets of adipocytes were visualized using an oil red O staining reagent (Sigma, Saint Louis, Mo., USA). Osteogenic differentiation was confirmed by staining calcium accumulation with alizarin red (Sigma, Saint Louis, Mo., USA), and chondrogenic differentiation was confirmed by an alcian blue staining (Sigma, Saint Louis, Mo., USA).
  • Immunophenotyping In order to confirm the characteristics of hMSCs, cells within five passages were stained with the cell surface markers CD29, CD73, CD90, CD105, CD34, CD45 (BD Pharmingen, Heidelberg, Germany), and CD44 (BD Biosciences, San Diego, Calif.). The expression of the cell surface markers was measured using a flow cytometer (BD FACS Canto ⁇ II), and hMSCs were identified as CD29/CD44/CD73/CD105-positive and CD34/CD45-negative cells.
  • a rotarod test was performed one day before hMSC transplantation and weekly for 4 weeks after hMSC transplantation, and a simple composite phenotype scoring system was performed once every four weeks after hMSC transplantation.
  • Rotarod test The rotarod test was used to evaluate motor coordination and balance according to the method disclosed in the prior literature (Zhang M J, Sun J J, Qian L, Liu Z, Zhang Z, Cao W, Li W, Xu Y: Human umbilical mesenchymal stem cells enhance the expression of neurotrophic factors and protect ataxic mice. Brain Res 2011, 1402:122-131). Experimental mice were carefully placed on a rotating rod weekly for 4 weeks. The rotation speed was increased linearly from 4 rpm to 40 rpm for 5 minutes, and the same speed (40 rpm) was maintained for 5 minutes. The time (latency) it takes for the mouse to lose balance and fall off the rod, i.e., the total time the mouse could remain on the rotating rod was recorded. In order to prevent muscle fatigue, each mouse was allowed to rest for 10 minutes between each experiment.
  • Simple composite phenotype scoring system In order to quantitatively analyze the severity of the disease in the LPS-induced cerebellar ataxia mouse model, a conventionally known scoring system was used in combination with the ledge test and the hindlimb clasping test (Guyenet S J, Furrer S A, Damian V M, Baughan T D, La Spada A R, Garden G A: A simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia. J Vis Exp 2010). The scoring system was done in 14-week-old mice, all tests were rated on a 0-3 point scale, and a composite phenotype score of 0-6 was added: A score of 0 means no relevant phenotype, and a score of 3 means the most severe symptom. Each test was performed three times.
  • Ledge test Motor imbalance and ataxia were evaluated through the ledge test. A score of 0 was applied when the mouse walked along a ledge without losing overall balance, a score of 1 was applied when the mouse walked in an unbalanced position along a ledge, a score of 2 was applied when the mouse impacted while walking along a ledge, and a score of 3 was applied when the mouse was unable to use its hindlimbs effectively.
  • Hindlimb clasping test This test was used as a marker of disease progression in a mouse model of cerebellar ataxia (Chou A H, Yeh T H, Ouyang P, Chen Y L, Chen S Y, Wang H L: Polyglutamine-expanded ataxin-3 causes cerebellar dysfunction of SCA3 transgenic mice by inducing transcriptional dysregulation. Neurobiol Dis 2008, 31:89-101).
  • a score of 0 was applied when all of the mouse's hindlimbs were consistently spread out from the abdomen, a score of 1 was applied when one hindlimb was retracted toward the abdomen for more than 50% of the measurement time, a score of 2 was applied when both hindlimbs were partially retracted toward the abdomen for more than 50% of the measurement time, and a score of 3 was applied when both hindlimbs were completely retracted toward the abdomen or touched the abdomen for more than 50% of the measurement time.
  • total cell lysates were prepared from cerebellar vermis for the mice injected with LPS and from the entire cerebellum for the mice injected with Ara-C.
  • the cerebellar vermis was isolated, and each tissue was homogenized with a protease inhibitor cocktail (1:100, Millipore, Burlington, Mass., USA) and a phosphatase inhibitor cocktail (1:100, Cell Signaling Technology) in a lysis buffer (58 mM Tris-HCl, pH 6.8; 10% glycerol; and 2% SDS).
  • the lysate was centrifuged, and then the protein concentration was quantified using a BCA kit (Bio-Rad Laboratories, Hercules, Calif., USA).
  • Proteins were separated using gel electrophoresis and then transferred onto a membrane using an electrophoresis transfer system (Bio-Rad Laboratories). The membrane was incubated overnight at 4° C. with the following primary antibodies: anti-Iba1 (anti-ionized calcium-binding adapter molecule 1, 1:1500, Wako, Osaka, Japan), anti-GFAP (anti-glial fibrillary acidic protein, 1:2000, Millipore, Billerica, Mass., USA), anti-TNF ⁇ (anti-tumor necrosis factor alpha, 1:1000, Abcam, Cambridge, UK), anti-IL-1 ⁇ (1 beta, 1:1000, Abcam, Cambridge, Mass., USA), anti-MCP-1 (anti-monocyte chemoattractant protein 1, 1:500, Abcam, Cambridge, UK), anti-MIP-1 ⁇ (anti-macrophage inflammatory protein 1 alpha, 1:1000, R&D Systems, Minneapolis, Minn., USA), anti-Cal-D28K (anti-calbindin-D-28K
  • anti-cleaved caspase-3 (1:1000, Cell Signaling, Beverly, Mass., USA), anti-caspase-3 (1:1000, Cell Signaling, Beverly, Mass., USA
  • anti-CD86 anti-cluster of differentiation 86, 1:1000, Invitrogen, Carlsbad, Calif., USA
  • anti-CD206 anti-cluster of differentiation 206, 1:1000, R&D Systems, Minneapolis, Minn., USA
  • anti-iNOS anti-inducible nitric oxide synthase, 1:1000, Abcam, Cambridge, UK
  • anti-IL-10 anti-interleukin 10, 1:1000, Abcam, Cambridge, Mass., USA
  • anti-TSG-6 anti-TNF ⁇ stimulated gene-6, 1:1000, GeneTex, Irvine, Calif., USA
  • anti-beta actin anti-(31:2000, Santa Cruz, Calif., USA).
  • LPS was directly injected into the cerebellum as an effective substance for inflammatory neurodegeneration.
  • Iba1 microglia
  • IL-1 ⁇ and TNF ⁇ inflammatory cytokines
  • FIG. 1 A **p ⁇ 0.01, *p ⁇ 0.05 vs. CON
  • the expression levels of gliacytes and inflammatory cytokines in mice treated with PBS were not different from those of the control group.
  • MIP-1 ⁇ and MCP-1 inflammatory chemokines
  • chemoattractants for inflammatory cells and mesenchymal stem cells were confirmed in the cerebellum injected with LPS.
  • FIG. 1 A the expression of MIP-1 ⁇ and MCP-1 in the cerebellum was significantly increased at 1 day after LPS injection compared to the control group (**p ⁇ 0.01 vs. CON) and decreased therefrom after 7 days.
  • the motility defect and Purkinje cell loss of the mouse were confirmed through the rotarod test.
  • the residence time on the rotating rod was significantly reduced at 1 week after LPS injection compared to the normal control group, and the decreased value was maintained for 4 weeks ( FIG. 1 B ; ***p ⁇ 0.001 2 weeks and 3 weeks after LPS injection vs. CON, **p ⁇ 0.01 4 weeks after LPS injection vs. CON).
  • calbindin protein was significantly reduced in the cerebellum on day 7 after LPS injection in the mice injected with LPS compared to the normal control group, indicating a loss of Purkinje cells ( FIG. 1 B ; ***p ⁇ 0.001 vs. CON).
  • Ara-C was intraperitoneally injected daily for 1 to 3 days after birth.
  • the structure of the cerebellum, neurons, and Purkinje cells were confirmed through histopathological staining. As a result, it was confirmed that the development of the cerebellum of the animals administered with Ara-C was inhibited, and it was confirmed that neurons and Purkinje cells of the cerebellum were not formed.
  • the disease animal model was evaluated through behavioral evaluation. As a result, it was confirmed that there was a behavioral imbalance compared to the control group, such as parallel, hanging, and gait ( FIG. 2 B ).
  • B6D2-Tg(Pcp2-SCA2)11Plt/J mouse was established by inserting and overexpressing the human SCA2 gene into the mouse Pcp2 promoter.
  • the secured B6D2-Tg(Pcp2-SCA2)11Plt/J mouse was an animal model established by fertilization of C57BL/6J ovum and DBA/2J sperm. Because the genetic background was not uniform, crossing was carried out until the fifth generation, which showed 94% genetic stability through repeated backcrossing with C57BL/6J mice.
  • the properties of the mesenchymal stem cell therapeutic agent isolated from the bone marrow were confirmed through analysis of shape, marker, and differentiation ability, and the functional properties of the stem cells were also confirmed through the secreted cytokines.
  • hMSCs were determined by the morphology of fibroblastoid, the expression pattern of hMSC-related surface markers, and differentiation possibility according to the criteria of the International Society for Cellular Therapy (ISCT).
  • ISCT International Society for Cellular Therapy
  • hMSCs isolated from the bone marrow the original shape of spindle-shaped fibroblast-shaped stem cells could be visually confirmed ( FIG. 4 A ).
  • the PDT of hMSCs for each passage was evaluated to confirm the proliferative capacity of stem cells. Although hMSCs at the early passage stage showed high proliferative capacity (37-51 hours), it was confirmed that the proliferative capacity of hMSCs was poor in the late passage stage (after Passage 7) ( FIG. 4 B ).
  • stem cells at the early stage were cultured in adipogenic medium, osteogenic medium and chondrogenic medium for 2 to 4 weeks to confirm differentiation into adipocytes, osteoblasts and chondrocytes.
  • the neurotropic factor was identified to identify the secreted protein of the mesenchymal stem cell therapeutic agent.
  • the expression of ANG, BDNF, and IGF which are closely related to neuroprotective factors and growth and differentiation of neurons, was confirmed. It is thought that the mesenchymal stem cell therapeutic agent may be beneficial for a neurodegenerative disease ( FIG. 4 E ).
  • mice in all groups showed a similar level of motor coordination, but after LPS administration, it was confirmed that the disease animal had gradually decreased motility compared to the control group.
  • the protective effect of a stem cell therapeutic agent capable of inhibiting and protecting the apoptosis of Purkinje cells induced by inflammatory response was confirmed.
  • the Purkinje cell marker Cal-D28K was reduced in the cerebellum of the disease animal, but it was confirmed that the expression of the marker was higher in a group administered with the mesenchymal stem cell therapeutic agent compared to the control group ( FIG. 5 E ).
  • M1-type microglia of CD86 were reduced and M2-type microglia of CD206 were increased ( FIG. 5 F ). This indicates that M1-type microglia are polarized into M2-type microglia related to tissue regeneration, and this can represent the secondary therapeutic improvement effect of stem cells.
  • stem cells were administered in the following four ways:
  • the cerebellar ataxia model (Ara-C) mice administered with Ara-C failed to maintain balance and fell off in the rotarod test compared to the normal control group (WT).
  • the Ara-C model mice were administered with hMSCs, there was a difference in the enhancement of motor performance capability according to the administration dose and method. Specifically, when the mice were administered with 2 ⁇ 10 4 cells, there was no significant difference compared to the mice which were not administered with the stem cells. When 2 ⁇ 10 4 cells were repeatedly administered and when 6 ⁇ 10 4 cells were administered once, the balance ability was gradually increased until 16 weeks of age, but after that, it was reduced again.
  • the open field test was performed.
  • stem cells were administered once or repeatedly at two concentrations (1 ⁇ 10 5 or 1 ⁇ 10 6 cells), and the results were compared. Specifically, stem cells at each concentration were administered once at 10 weeks of age or administered repeatedly 3 times at a 4-week interval, and then at 22 weeks of age, general motility was measured through the open field test. As a result, it was confirmed that the motility was significantly reduced in the cerebellar ataxia model (Ara-C) mice administered with Ara-C compared to the normal control group (WT), but the motility was enhanced after administration of stem cells.
  • the effect of improving motor activity was more pronounced when the stem cells were administered repeatedly than when the stem cells were administered once, and in both the single administration and the repeated administration, when the stem cells were administered at a concentration of 1 ⁇ 10 5 cells, the effect of improving motor activity was higher ( FIG. 6 C ).
  • the effect of the therapeutic agent was also confirmed by differences in the protein. After administration of mesenchymal stem cells at the concentration of 1 ⁇ 10 5 cells in the disease animal, it was confirmed that the expression of the neuron protein NeuN and the neuroblast and neuron progenitor cell marker DCX (doublecortin) was significantly increased. This is considered to be a therapeutic effect due to improvement of maturation disorder of the cerebellum ( FIG. 6 D ).
  • the mesenchymal stem cell therapeutic agent was administered once or repeatedly at two concentrations (1 ⁇ 10 5 or 1 ⁇ 10 6 cells), and the results were compared. Specifically, stem cells at each concentration were administered once at 10 weeks of age or administered repeatedly 3 times at a 4-week interval, and then at 22 weeks of age, the effect of improving behavioral disorders was measured through the composite phenotype scoring system.
  • the treatment method or the treatment composition using mesenchymal stem cells, according to the present invention has remarkable effects in reducing neuroinflammation, inhibiting M1 microglia, activating M2 microglia, inhibiting apoptosis of Purkinje cells, inhibiting death of neurons, improving motor ability, and the like, and therefore may be effectively utilized in alleviating and treating neurodegenerative diseases including cerebellar ataxia and multiple system atrophy, and thus has high industrial applicability.

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